Mesenchymal stem cells derived from human teeth and bone marrow have been characterized by many research groups, but demonstrate inconsistent cellular phenotypes or functions, partly because of differences in culture methodology. Therefore, our aims were to resolve these inconsistencies and discuss the potential uses of these cells in research/clinical applications. We isolated and characterized dental stem cells (DSCs) from the dental pulp, periodontal ligament, apical papilla (APSCs) and dental follicle (DFSCs) of mature and immature teeth, along with bone marrow-derived stem cells (BMSCs) from the iliac crest. We compared the clonogenic and proliferative potentials of these cells in terms of colony-forming efficiency, proliferation potential, population doubling time and cell cycle. All DSCs, particularly APSCs and DFSCs, possessed greater proliferative potential than BMSCs. All stem cells expressed typical mesenchymal and embryonic markers, and developed alizarin red-positive mineralization nodules and Oil red O-positive lipid droplets when cultured in osteogenic and adipogenic media, respectively. Immunocytochemistry revealed that all stem cells developed neuronal markers when cultured in a control medium without neural inductive supplements. After 7 days of neurogenic culture, the differentiated cells showed a transition from fibroblast-like to neuron-like cell bodies with long processes, suggesting that the stem cells differentiated into mature neurons. Karyotyping confirmed that the stem cells maintained a normal karyotype and were chromosomally stable. Our results provide new insights into the physiological properties of stem cells with a normal karyotype and indicate that DSCs are appropriate for basic research and clinical applications.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for twodimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth.

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1007/s13577-017-0161-2) contains supplementary material, which is available to authorized users.

In recent decades, de-differentiated fat cells (DFAT cells) have emerged in regenerative medicine because of their trans-differentiation capability and the fact that their characteristics are similar to bone marrow mesenchymal stem cells. Even so, there is no evidence to support the osteogenic induction using DFAT cells in periodontal regeneration and also the co-culture system. Consequently, this study sought to evaluate the DFAT cells co-culture with periodontal ligament stem cells (PDLSCs) in vitro in terms of gene expression by comparing runt-related transcription factor 2 (RUNX2) and Peroxisome proliferator-activated receptor gamma 2 (PPARγ2) genes. We isolated DFAT cells from mature adipocytes and compared proliferation with PDLSCs. After co-culture with PDLSCs, we analyzed transcriptional activity implying by DNA methylation in all adipogenic gene promoters using combined bisulfite restriction analysis. We compared gene expression in RUNX2 gene with the PPARγ2 gene using quantitative RT-PCR. After being sub-cultured, DFAT cells demonstrated morphology similar to fibroblast-like cells. At the same time, PDLSCs established all stem cell characteristics. Interestingly, the co-culture system attenuated proliferation while enhancing osteogenic gene expression in RUNX2 gene. Using the co-culture system, DFAT cells could trans-differentiate into osteogenic lineage enhancing, but conversely, their adipogenic characteristic diminished. Therefore, DFAT cells and the co-culture system might be a novel cell-based therapy for promoting osteogenic differentiation in periodontal regeneration.